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目的:探讨Toll样受体4(TLR4)对脂多糖(LPS)促进人肺癌SPCA1细胞增殖的影响。方法:采用LPS(10μg/ml)刺激细胞,模拟慢性炎症的细胞微环境。将靶向TLR4基因的小干扰RNA(TLR4-siRNA)或阴性对照(NC-siRNA)通过脂质体介导转染人肺癌SPCA1细胞,24 h后加入10μg/ml LPS。按转染siRNA和加入LPS情况,实验分为不做任何处理的Control组、NC+10LPS组以及TLR4-siRNA+10LPS组。采用Real-time PCR和流式细胞术检测SPCA1细胞中TLR4 mRNA及蛋白的表达情况;采用CCK-8法和平板克隆形成实验检测细胞增殖能力,流式细胞术检测分析细胞周期分布情况。结果:与NC+10LPS组相比较,TLR4-siRNA+10LPS组细胞中TLR4 mRNA及蛋白的表达水平显著下降(P<0.01);与Control组和NC+10LPS组相比较,TLR4-siRNA+10LPS组SPCA1细胞的增殖明显减缓(P<0.01),TLR4-siRNA+10LPS组细胞克隆形成能力明显降低[(4.50±1.89)vs(13.33±1.81)、(15.75±1.25)个,P<0.01];TLR4-siRNA+10LPS组细胞周期阻滞于G0/G1期[(61.55±0.55)%vs(53.59±1.59)%、(51.72±0.77)%,P<0.01]。结论:TLR4-siRNA能有效沉默SPCA1细胞中TLR4的表达,能够阻滞细胞的生长,抑制细胞的增殖。
Objective: To investigate the effect of Toll-like receptor 4 (TLR4) on the proliferation of human lung cancer SPCA1 cells induced by lipopolysaccharide (LPS). Methods: Cells were stimulated with LPS (10μg / ml) to simulate the cellular microenvironment of chronic inflammation. Small interfering RNA (TLR4-siRNA) or negative control (NC-siRNA) targeting the TLR4 gene was transfected into human lung cancer SPCA1 cells by lipofectamine. After 24 h, 10 μg / ml LPS was added. According to the transfection of siRNA and LPS, the experiment was divided into Control group, NC + 10LPS group and TLR4-siRNA + 10LPS group without any treatment. Real-time PCR and flow cytometry were used to detect the expression of TLR4 mRNA and protein in SPCA1 cells. Cell proliferation was measured by CCK-8 assay and plate clone formation assay. Cell cycle distribution was analyzed by flow cytometry. Results: Compared with NC + 10LPS group, the expression of TLR4 mRNA and protein in TLR4-siRNA + 10LPS group was significantly decreased (P <0.01). Compared with NC + 10LPS group, TLR4- The proliferation of SPCA1 cells was significantly slowed down (P <0.01). The ability of cell cloning in TLR4-siRNA + 10 LPS group was significantly lower than that in control group (4.50 ± 1.89 vs 13.33 ± 1.81, (15.75 ± 1.25), P <0.01] The cell cycle arrest in-siRNA + 10 LPS group was (61.55 ± 0.55)% vs (53.59 ± 1.59)%, (51.72 ± 0.77)%, P <0.01, respectively. Conclusion: TLR4-siRNA can effectively silence the expression of TLR4 in SPCA1 cells, which can block the cell growth and inhibit the proliferation of cells.